The extracellular calcium-sensing receptor (CaR) binds and signals in response to Ca++ ions. By coordinating parathyroid hormone (PTH) secretion and renal calcium handling in response to fluctuations in extracellular Ca++, the CaR is critical to maintaining systemic calcium homeostasis. This receptor is also expressed in breast tissue and in breast cancers. We have found that the CaR is highly expressed in the normal mouse mammary gland during lactation, but is not expressed during pregnancy. Activation of the CaR inhibits the production of parathyroid hormone-related protein (PTHrP) by lactating mammary epithelial cells (MECs) both in vitro and in vivo. Furthermore, we have shown that CaR signaling promotes the transepithelial transport of calcium and water into milk. Based on these data, we propose a new paradigm: that the mammary gland becomes a calcium-sensing organ during lactation and actively participates in the regulation of systemic calcium and bone homeostasis. We propose that the CaR increases PTHrP secretion when calcium delivery to the mammary gland falls in order to increase maternal bone resorption, protect against maternal hypocalcemia and restore the supply of calcium for milk production. We also propose that the mammary CaR matches the transport of calcium into milk with the maternal supply of calcium. This would protect against severe hypocaclemia in the mother by reducing demand if supplies of calcium became limiting. Finally, given the loss of CaR expression during pregnancy, a time of intense MEC proliferation, we hypothesize that CaR signaling inhibits MEC proliferation and promotes MEC differentiation. In order to test these hypotheses we propose 4 Specific Aims. Aim 1 will examine mammary PTHrP production, water and calcium transport and maternal bone and mineral metabolism in a mouse model of mammary-specific CaR gene deletion. Aim 2 will examine the regulation of the plasma membrane Ca++/ATPase 2 (PMCA2) by CaR signaling in MECs. Aim 3 will examine the signaling pathways that mediate the mammary CaR's regulation of PTHrP production. Aim 4 will examine the effects of CaR signaling on MEC proliferation by expressing an activated mutant of the CaR in the mammary gland during pregnancy in transgenic mice. We believe that these studies will deepen our understanding of the physiology of the CaR and will contribute to an understanding of both osteoporosis and bone metastases in patients with breast cancer.